Etiologies of Persistent Aminotransferase Elevations in Chronic Hepatitis B Patients Treated with Nucleos(t)ide Analogs

Background/Aims: Recent studies revealed that patients with persistent aminotransferase elevations after antiviral treatment had higher risk of hepatic events; yet its underlying causes remain unclear. Our study aimed to investigate the etiologies of persistent aminotransferase elevations in patients treated with nucleos(t)ide analogs (NAs). Materials and Methods: A retrospective study was conducted on chronic hepatitis B (CHB) patients who had been receiving NA treatment for over a year and had an aminotransferase level greater than 40 IU/mL (more than twice, with a 3-month interval) and subsequently underwent a liver biopsy. Results: The study group included 46 patients (34 males) with a mean age of 44.8 ± 20.3 years (range: 24-71 years).The average duration of NA therapy was 3.7 years (1.1-10.6 years). The etiologies of persistant transaminase elevation were categorized into 4 groups: patients with low hepatitis B virus (HBV) viral load (LVL, n = 11); concurrent non-alcoholic fatty liver disease (NAFLD, n = 12); concurrent other liver diseases (OLD, n = 12); and unknown liver dysfunction (ULD, n = 11). The proportion of G ≥ 2 inflammation was significantly higher in the LVL group (90.9%) compared to NAFLD (33.3%), OLD (50%), and ULD (27.2%) groups (P = .012). The hepatitis B e-antigen (HBeAg)-positive group exhibited a younger age (34.5 ± 10.2 vs. 48.1 ± 9.4 years, P < .001), a lower proportion of fibrosis F ≥ 2 (36.3% vs. 77.1%, P = .012), and a higher prevalence of detectable HBV DNA (54.5% vs.14.2%, P = .00632) compared to the HBeAg-negative group. Conclusion: The etiology of persistent aminotransferase elevations in CHB patients undergoing NAs treatment warrants investigation. Besides the commonly observed NAFLD and low HBV viral load, concurrent presence of other liver diseases requires elucidation.The proportion of G≥2 inflammation was higher in the LVL group.


INTRODUCTION
Hepatitis B virus (HBV) infection is a major cause of cirrhosis and hepatocellular carcinoma (HCC), both of which endanger global health and impose a significant economic burden.[3][4] The biochemical response is one of the important indicators for judging the effect of NA treatment in chronic hepatitis B (CHB) patients.Early alanine aminotransferase (ALT) or aspartate aminotransferase (AST) normalization after NA treatment has been shown to reduce the development of cirrhosis and HCC. 5 However, even after 1 year of treatment with tenofovir disoproxil fumarate (TDF) or entecavir (ETV), 2 first-line therapies with strong barriers to resistance, the rates of ALT normalization were reported to be 68%-76% and 67%-78%, respectively. 5,6here is limited information on the possible causes of the persistent aminotransferase elevations in CHB patients treated with NAs.
4][5][6][7] The etiologies of persistent aminotransferase elevations in CHB patients with complete virus suppression may be associated with other liver injuries: most commonly concurrent alcoholic consumption or non-alcoholic fatty liver disease (NAFLD); co-infection with hepatitis C virus (HCV), 8 hepatitis D virus (HDV), 9 hepatitis E virus (HEV), Human Immunodeficiency Virus (HIV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV), and concurrent rare liver diseases such as autoimmune liver disease, metabolic or hereditary liver diseases. 10Because these rare liver diseases are challenging to diagnose using standard laboratory tests, liver pathology or even genetic information is frequently required.However, few people had a liver biopsy, which frequently resulted in clinical misdiagnosis.On the other hand, persistently elevated serum aminotransferase levels increase the risk of fulminant hepatitis, cirrhosis, and HCC. 11Early detection of the cause and treatment are especially crucial to slow down or prevent the progression of the disease and improve the long-term prognosis of patients.
Herein, we retrospectively analyzed our CHB patients who had been taking NAs for more than a year and had ALT levels higher than 40IU/mL (more than twice, with a 3-month interval) and underwent a liver biopsy to promote early identification of the etiology of abnormal ALT levels and initiate timely treatment.

MATERIALS AND METHODS Study Population
The study group included CHB patients who underwent liver biopsy at the Second Hospital of Nanjing between January 2018 and December 2020 and met the following criteria: those who had been taking NAs for more than a year and had an ALT level greater than 40 IU/mL (more than twice, with a 3-month interval).Exclusion criteria included co-infection with HCV, HDV, HEV, HIV, EBV, or CMV; alcoholism (20 g/day), history of HCC, current interferon therapy, use of NAs for chemoprevention, and poor treatment compliance of patients.The relevant guidelines were used to make the diagnoses of autoimmune hepatitis (AIH), 12 primary biliary cholangitis (PBC), 13 druginduced liver injury (DILI), 14 progressive familial intrahepatic cholestasis (PFIC), 15 hemochromatosis, 16 idiopathic portal hypertension (IPH), Budd-Chiari syndrome (BCS), and congenital hepatic fibrosis (CHF). 17The assessment of medication adherence involved the utilization of a questionnaire in conjunction with the hospital's medication prescribing records.
This was a retrospective observational study, and the reported data was analyzed anonymously.All participants provided informed consent.The study protocol was approved by the Medical Ethics Committee of the Second Hospital of Nanjing (2018-LY-kt001, date: January 5, 2018).

Laboratory Methods
A blood routine was performed by the blood cell analyzer of Shenzhen Maiduan Company (BC-3000).The biochemical indexes were detected by OLYMPUS AU2700 automatic biochemical analyzer.Hepatitis B serologic tests were detected by Abbott's automatic chemiluminescence analyzer.HBV DNA was analyzed by StepOnePlus polymerase chain reaction (PCR) fluorescence analyzer (lower detection limit at 20 IU/mL; Roche Diagnostic System Inc, Branchburg, NJ, USA).Drug-resistant mutations were detected by the American ABI 3730XI sequencer.The serum immunoglobulin and its matching reagents were detected by the Deling BNII plasma protein analyzer (SIEMENS, Germany).Autoimmune antibodies were detected using an indirect immunofluorescence assay in conjunction with Western blot, with kits provided by Germany (EUROIMMUN) Medical Laboratory Diagnostics Co, LTD.

Liver Biopsy and Histopathology Assessment
All patients had an ultrasound-guided liver biopsy with a 16G needle using the US-made Bard automatic adjustable biopsy device.The liver tissue obtained must have a length greater than 2 cm and include a minimum of 11 portal areas.After fixation, embedding, and continuous sections, hematoxylin and eosin (H&E), and Masson staining of the reticular fiber was performed on liver tissue samples, or special staining was performed under specific conditions.Liver samples were evaluated in a blinded manner by 2 experienced liver pathologists.Liver tissue inflammation (G) and fibrosis (F) were graded according to The METAVIR scoring system, and steatosis was evaluated using the NAS scoring system. 18A score of greater than or equal to 5 correlated with the diagnosis of NASH.Likewise, scores less than 3 correlated with ''non-NASH'' and scores of 3 or 4 were regarded as borderline.When pathological changes from 2 diseases coexisted, abnormal ALT caused by pathological changes was regarded as the final diagnosis.

Statistical Analysis
The measurement data was expressed as mean ± SD (X ± SD), and the categorical variable data was compared by the chi-squared test or two-tailed Fisher's exact test.

DISCUSSION
According to liver histopathology, the etiology of persistent aminotransferase elevations was low HBV viral load, concurrent NAFLD, other liver diseases, and unknown.
The incidence of cirrhosis and HCC was higher in the partial virological response group compared to the virological response group during NA treatment. 4Despite the use of NAs with a high barrier to resistance [ETV, TDF, and tenofovir alafenamide (TAF)], some patients exhibited detectable HBV DNA even after several years of treatment, with the detection rates after 1, 2, and 3 years ranging from 7%-33%, 9%-25%, and 10%-17%, respectively. 3,4In this study, the average duration of NA treatment was 3.7 years (range: 1.1-10.6years), with an HBV DNA detection rate of 26.08%, surpassing prior study rates.This discrepancy might stem from earlier use of agents with low resistance barrier (LAM, telbivudine, or ADV), 3,4,19,20 reducing the efficacy of subsequent ETV or TDF rescue therapy, a notion supported by resistance testing.Additionally, the proportion of G ≥ 2 inflammation was 90.9% in the LVL group.This emphasizes the necessity for adjusting ongoing antiviral treatments and advocates for employing a sensitive PCR assay for HBV DNA detection in CHB patients receiving NAs.About 54.54% of patients in the ULD group had confirmed liver cirrhosis (F > 3), previous studies had shown liver cirrhosis as a risk factor for poor biochemical response in CHB patients. 3,4,19Further research is necessary to ascertain its role in this context.Concurrently, only 27.27% of the patients in this group had G ≥ 2 inflammation.Thus, the treatment remained unchanged and was still in the follow-up stage.
Non-alcoholic fatty liver disease was confirmed in 26.08% of the patients in this study, and it was one of the most common causes of persistent transaminase elevations during NA therapy. 10,21The proportion of G ≥ 2 inflammation was observed in 33.3% of the patients, lower than in the LVL group, owing to distinct pathological changes in CHB and NAFLD.CHB typically exhibits portal inflammation, while NAFLD manifests macrovesicular or mixed macrovesicular steatosis, primarily in the acinar zone 3, along with or without hepatocyte ballooning, mixed lobular inflammatory cell infiltration, and perisinusoidal fibrosis. 10Studies highlight significant interconnection between the HBV virus molecules and major NAFLD pathways; for instance, liver steatosis may relate to HBV-X protein and farnesoid X receptor. 22However, the impact of HBV infection on NAFLD occurrence or vice versa remains uncertain.While steatosis is presumed to accelerate fibrosis progression in chronic hepatitis C, the proportion of fibrosis F ≥ 2 in this group didn't significantly differ from the other 3 groups, indicating potential unrelatedness between fibrosis and fatty degeneration.Concurrently, diet control and exercise intervention were implemented alongside NA treatment.
In this study, 2 CHB patients with concurrent AIH were distinguished by the presence of moderate-to-severe interface hepatitis, portal plasma cell infiltrate, and hepatocyte rosette formation, emperipolesis; whereas CHB presented a mild interface hepatitis. 12The International Autoimmune Hepatitis Group diagnostic scoring system was used to confirm AIH. 8,12,23Two patients received immunosuppressive treatment with prednisolone while undergoing NA therapy, successfully achieving remission.Drug-induced liver injury is distinguished by explicit medication and supplement exposure, necrosis of centrilobular zone 3, and the absence of severe fibrosis or cirrhosis. 24otably, there were no flares in liver function following glucocorticoid withdrawal.
Iron deposition was identified in 2 cases via liver biopsy, indicated by Prussian blue staining levels of 3+ and 4+, separately.In CHB patients, iron metabolism disorders are associated with HBV infection.Serum iron overload can exacerbate liver iron deposition, inflammation, and progress liver fibrosis, leading to ALT elevations. 25Treatment included the administration of iron chelators alongside intermittent intravenous phlebotomies.One patient showed gradual improvement, while the other patient couldn't tolerate iron chelators and underwent a liver transplant in 2019.
IPH exhibited distinct hepatic lobular architecture, multiple dilated portal veins with thickened walls, penetrating the liver parenchyma, and the absence of fibrosis. 17The patient diagnosed with BCS presented hepatic segment inferior vena cava stenosis on CT scan and exhibited focal hepatic sinus dilatation and congestion within acinus zone 3 in the liver biopsy.She underwent a transjugular intrahepatic portosystemic shunt and was promptly  The HBeAg-negative CHB patients exhibited a significantly older age, a higher proportion of fibrosis F ≥ 2, and a lower prevalence of detectable HBV DNA compared to the HBeAg-positive patients.It is possible that HBeAgnegative CHB develops primarily from HBeAg-positive cases, which has a longer disease duration and long-term immune damage.[30] Data derived from real-life cohorts represent a patient spectrum broader than that found in randomized controlled trials, which frequently exclude individuals with multiple comorbidities.Consequently, these results hold greater relevance for routine clinical practice.Nevertheless, our study has a few limitations.First, only a subset of patients with persistently elevated ALT during antiviral therapy underwent liver biopsy.Secondly, the study was constrained by a small sample size.
In conclusion, patients with persistent aminotransferase elevations after NA therapy require immunological, histopathological, and even molecular genetic tests.These evaluations aim to identify potential concurrent liver diseases.Additionally, detection of HBV DNA using sensitive PCR testing is essential to exclude the possibility of low viremia.
Ethics Committee Approval: This study was approved by the Ethics Committee of the Second Hospital of Nanjing (approval number: 2018-LY-kt001; date: January 5,2018).
Informed Consent: Written informed consent was obtained from the patients who agreed to take part in the study.

Figure 1 .
Figure 1.Representative pictures of liver pathology: (A) A 27-year-old male's liver biopsy displayed fatty degeneration in 30% of the hepatocytes, along with scattered, marked ballooning, and spotty necrosis (H&E 30×).(B) A 54-year-old female exhibited ANA 1 : 320 and IgG 16.8 g/L; liver pathology indicated moderate-severe interface inflammation and plasma cell infiltration (H&E 20×).(C) A 47-year-old male had a dilated portal vein in two-thirds of the portal area, accompanied by a thickened wall, extending into the liver parenchyma (H&E 20×).(D and E) A 50-year-old male had ferritin levels of 2000 ng/mL and transferrin saturation at 95%; liver biopsy showed brown particles in the bile ducts and hepatocytes (D, H&E 20×),confirmed by Prussian staining positive (E, 20×).(F) In a 55-year-old male, liver pathology revealed diffuse focal necrosis in lobules, waxy Kupffer cells in acinus zone 3, and mild interface inflammation (H&E 20×).

Table 2 .
Comparison of Clinical and Pathological Features of CHB Patients in the Low Viral Load, Non-alcoholic Fatty Liver Disease, Other Liver Diseases, and Unknown Liver Dysfunction Groups ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; GGT, glutamyl transpeptidase; LVL, low viral load; NAFLD, non-alcoholic fatty liver disease; OLD, other liver diseases; PLT, platelets; ULD, unknown liver dysfunction; WBC, white blood cells.